Early Nutrition and the Establishment of Epigenotype at Metastable Epialleles

1. Early Nutrition and the Establishment of Epigenotype at Metastable Epialleles Rob Waterland Houston, Texas

2. The Waterland Lab Funding: NIH-NIDDK, March of Dimes, USDA

4. Metabolic Imprinting Adaptive responses to early nutrition Persistent effect Susceptibility limited to critical period of development Waterland & Garza Am J Clin Nutr 1999;69

5. Early Nutrition and DNA Methylation • Most cytosines within CpG dinucleotides are methylated • Tissue-specific patterns of CpG methylation are established during development • Methylation requires dietary methyl donors and cofactors • Mitotically heritable

6. Mammalian One Carbon Metabolism

7. Early Nutrition and DNA Methylation • Most cytosines within CpG dinucleotides are methylated • Tissue-specific patterns of CpG methylation are established during development • Methylation requires dietary methyl donors and cofactors • Mitotically heritable

8. Overall Hypothesis • Specific subsets of genes are especially sensitive to early nutritional influences on epigenetic regulation • Genomically imprinted genes • Metastable epialleles Waterland and Jirtle, Nutrition 2004

9. The Viable Yellow Agouti (Avy) Mouse

10. Maternal Methyl Donor Supplementation Affects Coat Color of Avy/a Offspring Waterland & Jirtle, Mol Cell Biol 2003

11. Supplementation Changes Coat Color by Increasing Avy Methylation Waterland & Jirtle, Mol Cell Biol 2003

12. Interpretation of Avy Experiment • Specific transposable elements induce epigenetic instability, allowing early diet to influence epigenotype • Transposable elements (SINEs, LINEs, etc.) comprise >40% of the human genome

13. Metastable Epiallele “An allele at which the epigenetic state can switch and establishment is a probabilistic event. Once established, the state is mitotically inherited.” Rakyan et al Trends in Genetics 2002 Viable yellow agouti Axin fused

14. IAP 6 7 The Axin Fused (AxinFu) Mouse • Axin regulates embryonic axis formation • Inhibitor of Wnt signaling pathway • AxinFu caused by IAP insertion into Axin intron 6 • Tail kink phenotype associated with expression of truncated transcript originating downstream of IAP • AxinFu methylation silences the mutant transcript Vasicek et al Genetics 1997

15. Methods • C57 (+/+) dams assigned to diets 2 weeks before mating with AxinFu/+ males • Control: NIH-31 • Supplemented: NIH-31 with extra • folic acid, B12, betaine and choline • Offspring rated for tail phenotype at age 21 d • AxinFu CpG methylation measured by bisulfite sequencing

16. Classification of Tail Phenotype None Slightly Kinky Kinky

17. Classification of Tail PhenotypeVery Kinky

18. Methods • C57 (+/+) dams assigned to diets 2 weeks before mating with AxinFu/+ males • Control: NIH-31 • Supplemented: NIH-31 with extra • folic acid, B12, betaine and choline • Offspring rated for tail phenotype at age 21 d • AxinFu CpG methylation measured by bisulfite sequencing with phosphor-imager quantitation

19. Characteristics of Offspring at Weaning Control Supplemented Number of litters 24 22 Wean weight (g)† 8.2 ± 0.1 7.8 ± 0.1 * Litter size† 6.0 ± 0.3 5.7 ± 0.4 Proportion AxinFu/+ pups per litter† 0.40 ± 0.04 0.56 ± 0.04 ** Total number of pups 144 125 Number of AxinFu/+ pups 56 68 † mean ± sem * P < 0.05 ** P < 0.01

20. AxinFu Methylation and Tail Phenotype Waterland et al, submitted

21. Supplementation Reduces Incidence of Tail Kinks in AxinFu/+ Offspring P=0.002 P=0.002 Waterland et al, submitted

22. Supplementation Does NOT Increase AxinFu Methylation in Liver P=0.05

23. AxinFu is Hypomethylated in Tail

24. Supplementation Prevents Tail Kinks by Reducing Tail-Specific Loss of Methylation P=0.05 P=0.009 Waterland et al, submitted

25. Significance • Similar to Avy, epigenetic metastability at AxinFu confers lability to early nutrition • Nutritional effects on DNA methylation during development may • Be tissue-specific • Occur at diverse ontogenic periods

26. Transgenerational Perpetuation of Obesity…By Epigenetic Mechanisms? • Avy mouse is an ideal model • Spontaneously hyperphagic • Compare offspring of lean a/a dams and obese Avy/a dams

27. Avy Transgenerational Obesity Study • Approach: • Maintain two separate populations of Avy/a mice on control (NIH-31) or methyl-supplemented diet (folic acid, vitamin B12, betaine, choline) • Pass the Avy allele through the female germline for several generations • Assess cumulative effects on body weight of Avy/a and a/a offspring

28. 121 56 Maternal Obesity Increases Body Weight at Weaning in F1 Offspring Offspring Body Weight Dams P<0.001 Avy/a a/a

29. Transgenerational Effect of Maternal Obesity:Wean Weight by Generation F3 vs. F1 P=0.004 62 131 121 Average for offspring of a/a dams

30. Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet a/a Offspring Control Diet

31. Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet a/a Offspring Avy/a Offspring Control Diet

32. Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet a/a Offspring Avy/a Offspring Control Diet Supplemented Diet

33. Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet a/a Offspring Avy/a Offspring EffectP value Generation <0.0001 Genotype <0.0001 Supplementation 0.002 N=498 total Control Diet Supplemented Diet

34. Genotype-Epigenotype-Diet Interaction:Can too many vitamins make us fat? Maternal Obesity Methyl Donor Supplementation Avy Genotype Epigenetic Alterations Affecting Weight Gain?

35. Same Genotype, Different Epigenotype

36. Bisulfite Sequencing at AxinFu Unaffected AxinFu Hypermethylated Affected AxinFu less methylated T C T C Site 1 2 3 4

37. Validation of Quantitative Bisulfite Sequencing: Exploit H19 DMR in C57BL/6 x Cast/Ei F1 Mice SacI digestion eliminates paternal allele MfeI digestion eliminates maternal allele Measure % methylation in known mixtures of SacI and MfeI digested DNA Waterland et al Hum Mol Genet 2006